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Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil... Venous thromboembolic (VTE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils; however, the molecular mechanisms of neutrophil death in VTE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil–platelet aggregate formation induced by tumor necrosis factor-α but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space. Introduction morbidity and mortality worldwide . Although it can Venous thromboembolism (VTE) is a complication of occur in any location of the venous system, it primarily multiple different medical conditions and a major cause of manifests clinically as deep vein thrombosis (DVT) or pulmonary embolism . Local microvascular venous thrombosis is common at sites of trauma or infections but occurs also in life-threatening systemic disease states as Correspondence: Daigo Nakazawa (daigo-na@med.hokudai.ac.jp)or H.-J. Anders (hjanders@med.uni-muenchen.de) disseminated intravascular coagulation . Endothelial dys- Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, function and the activation of coagulation factors in the Munich, Germany plasma are central elements in clot formation, but the clot Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, itself largely consists of cellular elements such as red Japan blood cells, platelets, and neutrophils all contributing to Full list of author information is available at the end of the article. These authors contributed equally: Daigo Nakazawa, Jyaysi Desai Edited by I. Lavrik © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 2 of 11 clot formation . Red blood cell-derived microvesicles or deoxynucleotidyl transferase dUTP nick-end labeling adenosine diphosphate (ADP) initiate thrombin genera- (TUNEL) staining. In addition, immunostaining of mye- 4,5 tion and platelet activation, respectively . The role of loperoxidase (MPO) and citrulinated histone-3 (CitH3), as platelets in VTE is less prominent than in arterial well as RIPK3 and phosphorylated MLKL, showed the thrombosis. Nevertheless, thrombocytosis has been presence of NETs with a suggestive involvement of MLKL attributed as risk factor for VTE . Pathogens and danger- activation (Fig. 1a). Next, we assessed the same parameters in a mouse model of inferior vena cava (IVC) thrombosis. associated molecular patterns (DAMPs) stimulate neu- trophils to activate the clotting system, an interaction IVC thrombus was induced in wild-type (C57BL/6N) male referred to as immunothrombosis . Neutrophils, them- mice by ligation of IVC below the left renal vein without selves, contribute to clot formation by releasing neu- manipulating the side branches. At 72 h after surgery, trophil extracellular traps (NETs), that is, networks thrombi developed in the IVC, in which infiltrated Ly6b consisting of extracellular chromatin, cytoplasmic, and leukocytes showed high expression of RIPK3-MLKL, granular proteins as well as histones that elicit immu- CitH3, and TUNEL positivity (Fig. 1b). Therefore, we nostimulatory and cytotoxic effects on microvascular conclude that IVC thrombi of human and mouse stains endothelial cells . Indeed, netting neutrophil, monocytes, positive for markers of necroptosis, NETs, and cell death. and platelets cooperate to initiate and propagate VTE . Currently, little is known about the molecular mechan- Pharmacological RIPK1 inhibition reduces clot size in isms of VTE-related NET formation. It is shown that murine IVC thrombosis platelets release high mobility group protein B1 To assess whether necroptosis signaling contributes to 10,11 (HMGB1), which indeed triggers NET formation ; clot formation, we evaluated the effect of pharmacological however, the execution pathway of neutrophil death and inhibition of necroptosis with the RIPK1 stabilizer chromatin release in this context remains unknown. necrostatin-1s (Nec1s) in the aforementioned IVC ligation Recently, receptor-interacting protein kinase-3 (RIPK3), a venous thrombosis model. Wild-type mice were pre- protein involved in inflammation as well as regulated treated prior to IVC ligation with Nec1s. Macroscopic necrosis , has been reported to promote platelet activation findings revealed that Nec1s treatment significantly in arterial thrombosis .Interestingly,RIPK3 is also reduced clot formation (measured as thrombus weight) expressed in neutrophils and contributes to crystal-induced after IVC ligation (Fig. 2). TUNEL staining showed that 14,15 and microparticle-induced NET formation ,aprocess Nec1s treatment reduced cell death inside thrombi compared to vehicle (Fig. 3a, b). Furthermore, the number associated with neutrophil death and is therefore named 16 + neutrophil necroptosis . Necroptosis is a regulated form of of infiltrating Ly6b granulocytes in thrombi of Nec1s- cell necrosis involving necrosome formation by RIPK3 and treated mice were significantly lower than in controls. The the pseudokinase mixed lineage kinase domain-like expression of RIPK3-MLKL and CitH3 in thrombi was 17–19 (MLKL) . Indeed, MLKL oligomers form pores into mainly co-localized with blood cells and the over- nuclear and plasma cell membranes facilitating cell necrosis expression was suppressed by Nec1s treatment and chromatin release into the extracellular space . Thus, (Fig. 3a–c). Flow cytometric analysis revealed increased + high MLKL-driven neutrophil necroptosis may contribute to CD11b Ly6G neutrophils in the peripheral blood of gout and other microparticle-triggered diseases involving IVC-ligated mice, which was attenuated by Nec1s treat- 16,21 NETs , but its role in VTE is speculative. Here, we ment (Supplemental Fig. 1a, b). These findings indicate hypothesized that MLKL-dependent neutrophil necroptosis that the mechanism of venous thrombus formation might may contribute to VTE, and thus employed specific involve programmed neutrophil cell death via RIPK3- antagonists and Mlkl-deficient mice to address this concept MLKL signaling and histone citrullination. Next, because experimentally in vitro and in vivo. monocytes and macrophages produce tissue factor lead- ing to the activation of pro-coagulant system, we eval- Results uated the infiltration of F4/80 macrophages in thrombi + high Inferior vena cava thrombi of human and mouse stains and the circulating CD11b Ly6G monocytes. Phar- positive for markers of necroptosis macological inhibition of RIPK1 reduced the number of To examine whether RIPK/MLKL-dependent necrop- infiltrating macrophages in thrombi and the circulating tosis is involved in thrombus formation, we performed monocytes (Fig. 3a–c and Supplemental Fig. 1a, c). Next, immunostaining of an autopsy sample of a patient with to examine the role of programmed necrosis-related inferior vena cava thrombus due to renal cell carcinoma. DAMPs during DVT formation, we measured serum Hematoxylin and eosin (H&E) staining showed that infil- histone–DNA complexes by sandwich enzyme-linked trating leukocytes were present in the thrombus along immunosorbent assay (ELISA). We observed that Nec1s with CD61 platelets and fibrinogen. The presence of dead treatment showed a trend toward a reduced titer of cells inside the thrombus was identified by terminal histone–DNA complexes (Supplemental Fig. 1d). These Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 3 of 11 Fig. 1 Programmed necrosis contributes to thrombus formation in human and mouse. a Paraffin-embedded sections of inferior vena cava (IVC) thrombus of patient with renal cell carcinoma (upper left panel). H&E staining shows leukocyte infiltration into the thrombus (upper right). Immunohistochemistry for CD61 and fibrinogen (lower panel). TUNEL, myeloperoxidase (MPO), receptor-interacting protein kinase-3 (RIPK3), and phosphorylated mixed lineage kinase-like (pMLKL) positive blood cells were detected in the thrombus. Scale bar = 500 μm. b Thrombus of mouse IVC ligation model. From the left panel, the staining shows TUNEL, Ly6b, RIPK3, MLKL, citrullinated histone-3 (CitH3). Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm) data imply that necroptotic neutrophils and NET-derived Mlkl deficiency reduces clot size in IVC thrombosis DAMPs could induce further recruitment of immune cells To validate the involvement of necroptosis and also to to the forming clot. Thus, blood cells play a critical role in avoid potential drug off-target effects, we applied a the development of DVT and RIPK inhibition ameliorated genetic approach using Mlkl-deficient mice. We venous thrombosis possibly via the suppression of neu- observed that Mlkl deficiency significantly reduced clot trophil necroptosis. size upon IVC ligation 3 days after surgery in mice Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 4 of 11 Fig. 2 Necrostatin-1s (Nec1s) inhibits thrombus formation. The IVC of wild-type male mice (11–13 weeks old) was ligated under the anesthesia with pretreatment of vehicle (5% DMSO in PBS) or Nec1s, and all mice were sacrificed 3 days after the operation. a Left and right image show the thrombus in IVC ligation model with vehicle and Nec1s, respectively. Upper photos show macroscopic findings and lower figures show H&E staining. Scale bar = 1 mm. b The graph shows the thrombus size of sham-operated mice with vehicle (n = 3) or Nec1s (n = 4), and IVC-ligated mice with vehicle (n = 7) or Nec1s (n = 7). Data are mean ± SEM in each group. **p < 0.01 vs. respective control (Fig. 4a, b). Similar to Nec1s treatment, the areas of MLKL protein in neutrophils, PBMCs, and platelets TUNEL+ cells, Ly6b+ neutrophils, and F4/80+ mac- (Supplemental Fig. 3c). Next, we explored whether rophages in thrombi of IVC-ligated Mlkl-deficient mice thrombin-activated platelets induce neutrophil necrop- were reduced compared to wild-type mice (Fig. 5a–c). tosis via the RIPK signaling pathway in vitro. Although Furthermore, the expression of RIPK3 and CitH3 in neutrophils were not directly affected by the addition of thrombi of IVC-ligated Mlkl-deficient mice was sig- thrombin and non-activated platelets, thrombin- nificantly lower compared to wild-type mice (Fig. 5a–c). activated platelets stimulated neutrophils to undergo In addition, Mlkl deficiency resulted in less circulating NET formation with high expression of CitH3, RIPK3, neutrophils, monocytes, and serum histone–DNA and MLKL and with lactate dehydrogenase (LDH) complexes after IVC ligation. There was no difference release, the latter indicating neutrophil death (Fig. 6a–e). between groups at baseline (before surgery) in the NET formation and LDH release were both suppressed number of neutrophils, monocytes, DAMPs, or bleeding by pretreatment with Nec1s (Fig. 6a–e). Considering the time (Supplemental Fig. 2a–d). Taken together, Mlkl presence of blood cells with phosphorylated MLKL in deficiency reduces clot size in IVC thrombosis, possibly human thrombus (Fig. 1a), the dead neutrophils undergo by abrogating MLKL-dependent necroptosis of blood necroptosis. To verify that this process contributes to cells, especially neutrophils. granulocyte–platelet aggregation as a central mechanism of clot formation, whole blood was incubated with tumor Activated platelets induce neutrophil necroptosis and necrosis factor-α (TNFα)/zVAD known as typical indu- neutrophil–platelet aggregation cer of necroptosis. As a readout granulocyte–platelet Because blood cell necroptosis and NET formation aggregation was analyzed by flow cytometric analysis were detected in thrombi of IVC-ligated mice, we using CD61 (platelet) and CD66 (granulocyte) markers. questioned which blood cells underwent necroptosis, TNFα/zVAD induced granulocyte–platelet interaction and how NETs are induced during thrombus formation? and pretreatment with the RIPK1 inhibitor Nec1s or the We firstexaminedthe expression of RIPK3and MLKL in MLKL inhibitor necrosulfamide (NSA) inhibited this human neutrophils, peripheral blood mononuclear cells process (Fig. 7a, b). Furthermore, as a second and more (PBMCs), and platelets by immunofluorescence staining physiological inducer thrombin triggered the same as well as immunoblotting. Immunostaining revealed interaction of granulocytes and platelets, which was that neutrophils and platelets both express RIPK3 and rescued by Nec1s and NSA pretreatment (Fig. 7c). MLKL (Supplemental Fig. 3a, b). In addition, immuno- Finally, we examined whether platelets themselves blotting analysis showed the presence of RIPK3 and undergo necroptosis by thrombin stimulation. The Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 5 of 11 Fig. 3 Nec1s suppresses necroptosis and NET-related signaling pathway during thrombus formation. a Representative figures of IVC-ligated mice with pretreatment of vehicle and b Nec1s. From left panel, the staining shows TUNEL, Ly6b, RIPK3, MLKL, CitH3, and F4/80. Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm). c Quantification of positive area of each staining. Data are mean ± SEM in each group. *p < 0.05 vs. respective control platelet death and activation in vitro was examined by Discussion flow cytometry using annexin V and P-selectin as acti- We had hypothesized that MLKL-dependent neutrophil vation markers, respectively. TNFα/zVAD-stimulated necroptosis would contribute to VTE and employed specific human platelets up-regulated annexin V and P-selectin; antagonists and Mlkl-deficient mice to address this concept however, these phenomena were not inhibited by experimentally both in vitro and in vivo. We found the Nec1s and NSA (Supplemental Fig. 4a). Similarly, essential mediators of necroptosis, RIPK3 and MLKL, to be thrombin up-regulated annexin V and P-selectin present in IVC thrombi of humans and mice. Interfering with expression in platelets, whereas Nec1s or NSA had no necroptosis, either with the specific antagonist Nec1s or with inhibitory effect (Supplemental Fig. 4b). Taken together, genetic deletion of Mlkl, partially protected mice from IVC activated platelets induce neutrophil necroptosis and ligation-induced venous thrombosis. Mechanistically, neutrophil–platelet aggregation. thrombin-related platelet activation triggered neutrophil Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 6 of 11 Fig. 4 Genetic depletion of Mlkl reduces thrombus size in the IVC ligation model. a Macroscopic findings of thrombi in IVC-ligated wild-type −/− −/− (left) and Mlkl mice. Scale bar = 1 mm. b The graph shows the thrombus size of IVC-ligated wild-type (n = 7) and Mlkl mice (n = 7). Data are mean ± SEM in each group. *p < 0.05 vs. respective control death and neutrophil-platelet aggregation, which both could numerous other crystals and microparticles of different be reversed by specific necroptosis inhibitors. Platelet acti- sizes and shapes induce neutrophil necroptosis via RIPK1, 14,15 vation itself did not involve this pathway. We, therefore, RIPK3, and MLKL . Neutrophil necroptosis has also conclude that in VTE activated platelets induce neutrophil been observed in other conditions including exposure to necroptosis, a process generating the release of chromatin granulocyte–macrophage colony-stimulating factor fol- and DAMPs that contribute to clot formation (Fig. 8). lowed by the ligation of adhesion receptors such as CD44, NETs form predominantly during the organizing stage of CD11b, CD18, or CD15 . Our data add thrombin-activated VTE development . NETs are released by neutrophils platelets to the potential triggers of neutrophil necroptosis, present in the lesion, and several studies identified platelet- although the precise outside-in signaling mechanism 9–11,23 derived HMGB1 as a trigger for NET formation remains to be determined. We also do not claim that the .Lytic proteases, histones, and DAMPs released along with NETs process of neutrophil necroptosis is identical to that of NET certainly contribute to the local and systemic inflammation release or what has been called “NETosis.” However, the 2,8,24,25 associated with VTE . However, the sticky DNA itself consequence of plasma cell rupture in neutrophil necrosis is seems to be an essential component of the clot and the same, that is, sticky NET-like chromatin immobilizes synergizes with the fibrin mesh to retain red blood cells . adjacent particles, which in VTE are red blood cells, pla- Indeed, endogenous DNases counterbalance this phenom- telets, and the fibrinogen mesh . enon in conceptually similar manner to plasmin that Interestingly, we found that platelets also express RIPK3 degrades the fibrin mesh . There is an ongoing debate and MLKL. These are ubiquitous cytoplasmic proteins, whether NET release is necessarily associated with neu- which are obviously shed from megakaryocytes during trophil death or not. During host defense neutrophils have platelet formation. Although platelet necrosis is known to been shown to continue migrating also after NET release, be regulated by mitochondrial effect with calcium reflux 16,27 31 but this has not been observed in other disease settings . and ATP depletion , how necroptosis contributes to the For example, in gout the exposure to urate crystals triggers platelet death remains unknown. It has been reported that crystal–NET aggregates involving lytic neutrophil death and deletion of RIPK3 from megakaryocytes and platelets causes production of a sticky creamy mass of NETs, dead a marked defect in platelet aggregation and attenuates 28,29 neutrophils, and crystals, called the gouty tophus .In dense granule secretion in response to thrombin or a VTE the process and results are conceptually similar, thromboxane A2 analog in vitro, and delay vascular although the additional presence of blood components such occlusion time in a mouse model of arterial thrombosis .It as platelets, red blood cells, and the fibrin mesh produce a should be noted that RIPK3 has promiscuous biological much higher consistency of clots vs. gouty tophi. Never- functions beyond necroptosis, for example, in apoptosis or theless, neutrophil death is essential in this process and interleukin-1-dependent or nuclear factor-κB-dependent 12 13 contributes to vascular occlusion, obstructing the blood inflammation , as well as platelet activation .Never- flow. Indeed, the interesting thing in the setting of VTE is theless, we did not find any evidence that MLKL inhibition that neutrophils undergo necroptosis, a form of regulated affects thrombin-induced platelet activation. Therefore, we cell necrosis . We recently described that urate as well as consider the VTE phenotype of Mlkl-deficient mice largely Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 7 of 11 Fig. 5 Mlkl gene deficiency suppresses necroptosis and NET-related signaling pathways during thrombus formation. From the left panel, −/− TUNEL, Ly6b, RIPK3, CitH3, and F4/80 staining. a Representative figures of IVC-ligated wild-type mice and b IVC-ligated Mlkl mice (scale bar = 1 mm). Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm). c Quantification of positive area of each staining. Data are mean ± SEM in each group. *p < 0.05 vs. respective control relate to the lack of neutrophil necroptosis. Unfortunately, necroptosis may interfere with clotting, which might be flox Mlkl mice were not accessible to us to study cell-type- explored for therapeutic purposes. specificdeletionofMLKL. In summary, in VTE activated platelets, and possibly Materials and methods other triggers, induce neutrophil necroptosis, a process Venous thrombosis model generating the release of chromatin and DAMPs One hundred percent flow obstruction of the IVC was that contribute to clot formation. Thus, inhibitors of induced in 12- to 13-week-old male wild-type C57BL/6N Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 8 of 11 Fig. 6 Activated platelets stimulate neutrophils leading to up-regulation of necroptosis-related and NET-related signaling molecules in vitro. a Upper, middle, and lower panel show co-culture images of neutrophils with non-activated platelets, neutrophils with thrombin-activated platelets, and Nec1s-treated neutrophils with thrombin-activated platelets. Immunofluorescent images show neutrophil elastase (NE): green; citrullinated histones H3 (CitH3): red; RIPK3: red; and MLKL: red. Scale bar = 50 μm. Quantification of CitH3 (b), RIPK3 (c), MLKL (d) area and LDH release of supernatant (e)in unstimulated neutrophils, thrombin-treated neutrophils, non-activated platelets, vehicle-treated neutrophils with thrombin-activated platelets, and Nec1s-treated neutrophils with thrombin-activated platelets. Data represent the mean ± SEM of three independent experiments and were analyzed using the paired t test. *p < 0.05 vs. respective control; **p < 0.01 vs. respective control mice (Charles River Laboratories, Sulzfeld, Germany) or sutures. Anesthesia was antagonized by subcutaneous −/− Mlkl mice under the maintenance of normal body injection of atipamezol 2.5 mg/kg and flumazenil 0.5 mg/ temperature by employing preoperative heat supply and kg and pain control was assured by regular subcutaneous online core body temperature recording as described . injections of buprenorphine 1 mg/kg every 8 h. Mice with Mice were anesthetized by intraperitoneal injection of surgical complications such as bleeding or injury of the medetomidine (0.5 mg/kg), midazolam (5 mg/kg), and IVC were excluded because these factors could possibly fentanyl (0.05 mg/kg) before median laparotomy was affect thrombus formation. Other groups of C57BL/6N performed to carefully expose and completely ligate the wild-type mice were treated with Nec1s (1.65 mg/kg, IVC using 7-0 prolene (ETHICON) exactly below the left intraperitoneally, Bio Vision, USA) or vehicle (10% renal vein without manipulating side branches. After dimethyl sulfoxide (DMSO) in phosphate-buffered saline ligation, the abdominal wall and skin were closed by (PBS)) 1 h before the surgery. All mice were sacrificed Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 9 of 11 Fig. 7 Nec1s and necrosulfonamide (NSA) inhibit aggregation between neutrophils and platelets in vitro. a Human whole blood was stimulated with TNFα/zVAD in the presence of vehicle, Nec1s, and NSA. Upper flow cytometry images show the platelet population gated by forward scatter (FSC)/sideward scatter (SCC). Lower images show neutrophil–platelet aggregates by CD61/CD66 gating and the aggregation ratio in TNFα/ zVAD-treated (b) and thrombin-treated (c) blood. Data represent the mean ± SEM of three independent experiments and were analyzed using the paired t test. *p < 0.05 vs. respective control 3 days after surgery and thrombus weight (without ves- Germany) was used to detect dying cells inside the sels) was measured as a primary endpoint of clot forma- thrombus following the manufacturer’s description. Positive tion. As a bleeding test 10- to 12-week-old male C57BL/ cells were quantified using the ImageJ software. −/− 6N wild-type or Mlkl mice were anesthetized using isofluorane. A 2 mm segment of the tail tip was cut Histological examination in human thrombus using a scalpel, and the tail was put in 37 °C PBS . Paraffin-embedded sections of a human IVC thrombus Bleeding time was recorded up to when bleeding had from a patient with renal cell carcinoma were stained with completely stopped. All animal-related procedures fulfilled H&E and immunohistochemistry was performed using the directive 2010/63/EU and were optimized in terms of the following primary antibodies rabbit anti-CD61 3R recommendations and approved by the local govern- (LifeSpan Biosciences, Inc. Seattle, WA, USA), rabbit mental authorities (ROB-55.2Vet-2532.Vet_02-17-54). anti-fibrinogen, rabbit anti-myeloperoxidase, rabbit anti- RIPK3, rabbit anti-phosphorylated MLKL (all from Histological examination Abcam, Cambridge, UK). Signal detected was performed Thrombi were embedded in paraffinand 3 μmsections using routine procedures as described . were deparaffinized and rehydrated as previously descri- bed . Sections were stained with H&E or prepared for In vitro experiments immunohistochemistry. A 0.3% H O was used for inhibi- Blood was obtained from healthy donors after providing 2 2 tion of endogenous peroxidase. Primary antibodies included written informed consent on forms approved by the ratanti-mouseLy6b(neutrophils, AbDSerotec,Oxford, “Ethikkommission der Medizinischen Fakultät der LMU” UK), rabbit anti-CitH3 (netting neutrophils, Abcam, Cam- and all experiments were performed in accordance with bridge, UK), rabbit anti-mouse RIPK3 (Abcam, Cambridge, their guidelines and regulations. Neutrophils were isolated UK), anti-mouse MLKL (kindly provided by Andreas Lin- using standard dextran sedimentation followed by 14,15 kermann, Dresden), and rat anti-mouse F4/80 (Serotec, Ficoll–Hypaque density centrifugation procedures . Oxford, UK) . TUNEL staining kit (Roche, Mannheim, For platelet isolation, blood was collected into sodium Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 10 of 11 Fig. 8 Schema of activated platelet-induced neutrophil necroptosis in DVT. During thrombus formation, RBCs initiate thrombin generation and platelet activation. The activated platelets affect neutrophils to induce neutrophil necroptosis via the phosphorylation of MLKL. Necroptotic neutrophil-derived extracellular chromatin can interact with fibrin mesh and activate endothelial cells, resulting in the aggravation of rigid clot formation as immunothrombosis. RBC red blood cell, pMLKL phosphorylated mixed lineage kinase domain-like citrate-coated tubes. Platelet-rich plasma was obtained by CD11b (BD Biosciences), and APC-CD45 (BioLegend) antibodies were used to identify circulating neutrophils centrifugation (200 rpm, 20 min). Neutrophils were sus- pended in RPMI (5 × 10 cells/well), and seeded onto and activated monocytes in peripheral blood. Mouse either eight-well micro-slides (Ibidi, Martinsried, Ger- plasma was analyzed for histone–nucleosome complexes many) or 96-well plates, and incubated in a 5% carbon by ELISA (Roche). Whole blood was analyzed by flow dioxide atmosphere at 37 °C for 30 min. Neutrophils were cytometry to quantify circulating immune cells . In vitro, pre-treated with Nec1s (100 µM, Enzo, Lörrach, Ger- human platelets and whole blood were used. Platelets many) or vehicle (1% DMSO in PBS) for 30 min and then were gated by APC-CD42b (BioLegend) and platelet stimulated with thrombin (0.05 U/ml, Merck Millipore, activation and death were determined by PerCP-CD62p Darmstadt, Germany), non-activated platelets, and (BioLegend) and FITC-Annexin V (BD Pharmingen), thrombin (0.05 U/ml for 3 min) activated platelets (1 × 10 respectively. Platelet–granulocyte aggregation was deter- cells/well). After 3-h incubation, the microslides were mined using anti-human FITC-CD61 and PE-CD66 fixed with 4% paraformaldehyde (PFA) and analyzed for (BioLegend) antibody in accordance with the manu- CitH3, RIPK3, and MLKL expression by immuno- facturer’s instructions. fluorescence staining. In 96 plates, neutrophil death was quantified by the LDH assay (Sigma Aldrich, Steinheim, Histone-nucleosome assay Germany) using neutrophil supernatants. To induce Serum histone was evaluated by histone–DNA com- neutrophil–platelet aggregation, human whole blood was plexes ELISA kit (Roche, Mannheim, Germany). stimulated by either the combination TNFα (200 ng/ml) and zVAD (20 µM) or 0.05 U/ml thrombin (for 10 min) Immunoblotting with or without pretreatment of Nec1s (100 µM) and Blood cells were also analyzed by standard immuno- necrosulfonamide (10 µM, Calbiochem). blotting. Cell pellets were lysed with RIPA buffer (Sigma, USA), the extracted proteins were separated by sodium Flow cytometric analysis dodecyl sulfate-polyacrylamide gel electrophoresis, and Flow cytometric analysis was performed on a FACS transferred to a polyvinylidene difluoride membrane. Calibur flow cytometer (BD Biosciences). In mouse Anti-β-actin, RIPK3, and MLKL antibodies (Abcam, UK) experiments, anti-mouse FITC-Ly6G, PerCP-Ly6C, PE- were used for detection of molecules expression. Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 11 of 11 Statistics 10. Stark, K. et al. Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood 128,2435–2449 (2016). Data are presented as mean ± SEM. Unpaired Student’s 11. Dyer, M. R. et al. Deep vein thrombosis in mice is regulated by platelet HMGB1 t test and one-way analysis of variance followed by Dun- through release of neutrophil-extracellular traps and DNA. Sci. Rep. 8,2068 nett’s post test were used for the comparison. A value of (2018). 12. Orozco, S. & Oberst, A. RIPK3 in cell death and inflammation: the good, the p < 0.05 was considered to indicate statistical significance. bad, and the ugly. Immunol. Rev. 277,102–112 (2017). 13. Zhang, Y. et al. Receptor-interacting protein kinase 3 promotes platelet acti- Acknowledgements vation and thrombosis. Proc.Natl. Acad.Sci.USA 114,2964–2969 (2017). The study was funded by a scholarship of the Alexander-von-Humboldt 14. Desai, J. et al. Particles of different sizes and shapes induce neutrophil Foundation to D.N. (1158708-STP2) and the Deutsche Forschungsgemeinschaft necroptosis followed by the release of neutrophil extracellular trap-like chro- (AN372/14-3 and 24-1 to H.-J.A., MU3906/1-1 to S.R.M. and STE2437/2-1 to S.S.) matin. Sci. Rep. 7, 15003 (2017). We thank James. M. Murphy and Warren Alexander, Cell Signaling and Cell 15. Desai, J. et al. PMA and crystal-induced neutrophil extracellular trap formation Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, involves RIPK1-RIPK3-MLKL signaling. Eur. J. Immunol. 46, 223–229 (2016). −/− Australia for supplying the Mlkl mice, Stefan Krautwald, University of Kiel, 16. Wang, X., Yousefi, S. & Simon, H. U. Necroptosis and neutrophil-associated Germany for shipment, and Andreas Linkermann, Dresden, for providing the disorders. Cell Death Dis. 9, 111 (2018). anti-MLKL antibody. 17. Linkermann, A. & Green,D.R. Necroptosis. N. Engl. J. 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Host DNases prevent vascular occlusion by neu- Received: 10 April 2018 Revised: 19 May 2018 Accepted: 3 June 2018 trophil extracellular traps. Science 358,1202–1206 (2017). 27. Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18,1386–1393 (2012). 28. Schauer, C. et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat. Med. 20,511–517 (2014). References 29. Desai, J., Steiger, S. & Anders, H. J. Molecular pathophysiology of gout. Trends 1. Stone, J. et al. Deep vein thrombosis: pathogenesis, diagnosis, and medical Mol. Med. 23,756–768 (2017). management. Cardiovasc. Diagn. Ther. 7,S276–S284 (2017). 30. Wang, X., He, Z., Liu, H., Yousefi, S. & Simon, H. U. Neutrophil necroptosis is 2. Wolberg, A. S. et al. Venous thrombosis. Nat. Rev. Dis. Prim. 1, 15006 (2015). triggered by ligation of adhesion molecules following GM-CSF priming. 3. Gando, S., Levi, M. & Toh, C. 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Autoimmun. 29,52–59 (2007). 819–835 (2012). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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Abstract

Venous thromboembolic (VTE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils; however, the molecular mechanisms of neutrophil death in VTE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil–platelet aggregate formation induced by tumor necrosis factor-α but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space. Introduction morbidity and mortality worldwide . Although it can Venous thromboembolism (VTE) is a complication of occur in any location of the venous system, it primarily multiple different medical conditions and a major cause of manifests clinically as deep vein thrombosis (DVT) or pulmonary embolism . Local microvascular venous thrombosis is common at sites of trauma or infections but occurs also in life-threatening systemic disease states as Correspondence: Daigo Nakazawa (daigo-na@med.hokudai.ac.jp)or H.-J. Anders (hjanders@med.uni-muenchen.de) disseminated intravascular coagulation . Endothelial dys- Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, function and the activation of coagulation factors in the Munich, Germany plasma are central elements in clot formation, but the clot Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, itself largely consists of cellular elements such as red Japan blood cells, platelets, and neutrophils all contributing to Full list of author information is available at the end of the article. These authors contributed equally: Daigo Nakazawa, Jyaysi Desai Edited by I. Lavrik © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 2 of 11 clot formation . Red blood cell-derived microvesicles or deoxynucleotidyl transferase dUTP nick-end labeling adenosine diphosphate (ADP) initiate thrombin genera- (TUNEL) staining. In addition, immunostaining of mye- 4,5 tion and platelet activation, respectively . The role of loperoxidase (MPO) and citrulinated histone-3 (CitH3), as platelets in VTE is less prominent than in arterial well as RIPK3 and phosphorylated MLKL, showed the thrombosis. Nevertheless, thrombocytosis has been presence of NETs with a suggestive involvement of MLKL attributed as risk factor for VTE . Pathogens and danger- activation (Fig. 1a). Next, we assessed the same parameters in a mouse model of inferior vena cava (IVC) thrombosis. associated molecular patterns (DAMPs) stimulate neu- trophils to activate the clotting system, an interaction IVC thrombus was induced in wild-type (C57BL/6N) male referred to as immunothrombosis . Neutrophils, them- mice by ligation of IVC below the left renal vein without selves, contribute to clot formation by releasing neu- manipulating the side branches. At 72 h after surgery, trophil extracellular traps (NETs), that is, networks thrombi developed in the IVC, in which infiltrated Ly6b consisting of extracellular chromatin, cytoplasmic, and leukocytes showed high expression of RIPK3-MLKL, granular proteins as well as histones that elicit immu- CitH3, and TUNEL positivity (Fig. 1b). Therefore, we nostimulatory and cytotoxic effects on microvascular conclude that IVC thrombi of human and mouse stains endothelial cells . Indeed, netting neutrophil, monocytes, positive for markers of necroptosis, NETs, and cell death. and platelets cooperate to initiate and propagate VTE . Currently, little is known about the molecular mechan- Pharmacological RIPK1 inhibition reduces clot size in isms of VTE-related NET formation. It is shown that murine IVC thrombosis platelets release high mobility group protein B1 To assess whether necroptosis signaling contributes to 10,11 (HMGB1), which indeed triggers NET formation ; clot formation, we evaluated the effect of pharmacological however, the execution pathway of neutrophil death and inhibition of necroptosis with the RIPK1 stabilizer chromatin release in this context remains unknown. necrostatin-1s (Nec1s) in the aforementioned IVC ligation Recently, receptor-interacting protein kinase-3 (RIPK3), a venous thrombosis model. Wild-type mice were pre- protein involved in inflammation as well as regulated treated prior to IVC ligation with Nec1s. Macroscopic necrosis , has been reported to promote platelet activation findings revealed that Nec1s treatment significantly in arterial thrombosis .Interestingly,RIPK3 is also reduced clot formation (measured as thrombus weight) expressed in neutrophils and contributes to crystal-induced after IVC ligation (Fig. 2). TUNEL staining showed that 14,15 and microparticle-induced NET formation ,aprocess Nec1s treatment reduced cell death inside thrombi compared to vehicle (Fig. 3a, b). Furthermore, the number associated with neutrophil death and is therefore named 16 + neutrophil necroptosis . Necroptosis is a regulated form of of infiltrating Ly6b granulocytes in thrombi of Nec1s- cell necrosis involving necrosome formation by RIPK3 and treated mice were significantly lower than in controls. The the pseudokinase mixed lineage kinase domain-like expression of RIPK3-MLKL and CitH3 in thrombi was 17–19 (MLKL) . Indeed, MLKL oligomers form pores into mainly co-localized with blood cells and the over- nuclear and plasma cell membranes facilitating cell necrosis expression was suppressed by Nec1s treatment and chromatin release into the extracellular space . Thus, (Fig. 3a–c). Flow cytometric analysis revealed increased + high MLKL-driven neutrophil necroptosis may contribute to CD11b Ly6G neutrophils in the peripheral blood of gout and other microparticle-triggered diseases involving IVC-ligated mice, which was attenuated by Nec1s treat- 16,21 NETs , but its role in VTE is speculative. Here, we ment (Supplemental Fig. 1a, b). These findings indicate hypothesized that MLKL-dependent neutrophil necroptosis that the mechanism of venous thrombus formation might may contribute to VTE, and thus employed specific involve programmed neutrophil cell death via RIPK3- antagonists and Mlkl-deficient mice to address this concept MLKL signaling and histone citrullination. Next, because experimentally in vitro and in vivo. monocytes and macrophages produce tissue factor lead- ing to the activation of pro-coagulant system, we eval- Results uated the infiltration of F4/80 macrophages in thrombi + high Inferior vena cava thrombi of human and mouse stains and the circulating CD11b Ly6G monocytes. Phar- positive for markers of necroptosis macological inhibition of RIPK1 reduced the number of To examine whether RIPK/MLKL-dependent necrop- infiltrating macrophages in thrombi and the circulating tosis is involved in thrombus formation, we performed monocytes (Fig. 3a–c and Supplemental Fig. 1a, c). Next, immunostaining of an autopsy sample of a patient with to examine the role of programmed necrosis-related inferior vena cava thrombus due to renal cell carcinoma. DAMPs during DVT formation, we measured serum Hematoxylin and eosin (H&E) staining showed that infil- histone–DNA complexes by sandwich enzyme-linked trating leukocytes were present in the thrombus along immunosorbent assay (ELISA). We observed that Nec1s with CD61 platelets and fibrinogen. The presence of dead treatment showed a trend toward a reduced titer of cells inside the thrombus was identified by terminal histone–DNA complexes (Supplemental Fig. 1d). These Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 3 of 11 Fig. 1 Programmed necrosis contributes to thrombus formation in human and mouse. a Paraffin-embedded sections of inferior vena cava (IVC) thrombus of patient with renal cell carcinoma (upper left panel). H&E staining shows leukocyte infiltration into the thrombus (upper right). Immunohistochemistry for CD61 and fibrinogen (lower panel). TUNEL, myeloperoxidase (MPO), receptor-interacting protein kinase-3 (RIPK3), and phosphorylated mixed lineage kinase-like (pMLKL) positive blood cells were detected in the thrombus. Scale bar = 500 μm. b Thrombus of mouse IVC ligation model. From the left panel, the staining shows TUNEL, Ly6b, RIPK3, MLKL, citrullinated histone-3 (CitH3). Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm) data imply that necroptotic neutrophils and NET-derived Mlkl deficiency reduces clot size in IVC thrombosis DAMPs could induce further recruitment of immune cells To validate the involvement of necroptosis and also to to the forming clot. Thus, blood cells play a critical role in avoid potential drug off-target effects, we applied a the development of DVT and RIPK inhibition ameliorated genetic approach using Mlkl-deficient mice. We venous thrombosis possibly via the suppression of neu- observed that Mlkl deficiency significantly reduced clot trophil necroptosis. size upon IVC ligation 3 days after surgery in mice Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 4 of 11 Fig. 2 Necrostatin-1s (Nec1s) inhibits thrombus formation. The IVC of wild-type male mice (11–13 weeks old) was ligated under the anesthesia with pretreatment of vehicle (5% DMSO in PBS) or Nec1s, and all mice were sacrificed 3 days after the operation. a Left and right image show the thrombus in IVC ligation model with vehicle and Nec1s, respectively. Upper photos show macroscopic findings and lower figures show H&E staining. Scale bar = 1 mm. b The graph shows the thrombus size of sham-operated mice with vehicle (n = 3) or Nec1s (n = 4), and IVC-ligated mice with vehicle (n = 7) or Nec1s (n = 7). Data are mean ± SEM in each group. **p < 0.01 vs. respective control (Fig. 4a, b). Similar to Nec1s treatment, the areas of MLKL protein in neutrophils, PBMCs, and platelets TUNEL+ cells, Ly6b+ neutrophils, and F4/80+ mac- (Supplemental Fig. 3c). Next, we explored whether rophages in thrombi of IVC-ligated Mlkl-deficient mice thrombin-activated platelets induce neutrophil necrop- were reduced compared to wild-type mice (Fig. 5a–c). tosis via the RIPK signaling pathway in vitro. Although Furthermore, the expression of RIPK3 and CitH3 in neutrophils were not directly affected by the addition of thrombi of IVC-ligated Mlkl-deficient mice was sig- thrombin and non-activated platelets, thrombin- nificantly lower compared to wild-type mice (Fig. 5a–c). activated platelets stimulated neutrophils to undergo In addition, Mlkl deficiency resulted in less circulating NET formation with high expression of CitH3, RIPK3, neutrophils, monocytes, and serum histone–DNA and MLKL and with lactate dehydrogenase (LDH) complexes after IVC ligation. There was no difference release, the latter indicating neutrophil death (Fig. 6a–e). between groups at baseline (before surgery) in the NET formation and LDH release were both suppressed number of neutrophils, monocytes, DAMPs, or bleeding by pretreatment with Nec1s (Fig. 6a–e). Considering the time (Supplemental Fig. 2a–d). Taken together, Mlkl presence of blood cells with phosphorylated MLKL in deficiency reduces clot size in IVC thrombosis, possibly human thrombus (Fig. 1a), the dead neutrophils undergo by abrogating MLKL-dependent necroptosis of blood necroptosis. To verify that this process contributes to cells, especially neutrophils. granulocyte–platelet aggregation as a central mechanism of clot formation, whole blood was incubated with tumor Activated platelets induce neutrophil necroptosis and necrosis factor-α (TNFα)/zVAD known as typical indu- neutrophil–platelet aggregation cer of necroptosis. As a readout granulocyte–platelet Because blood cell necroptosis and NET formation aggregation was analyzed by flow cytometric analysis were detected in thrombi of IVC-ligated mice, we using CD61 (platelet) and CD66 (granulocyte) markers. questioned which blood cells underwent necroptosis, TNFα/zVAD induced granulocyte–platelet interaction and how NETs are induced during thrombus formation? and pretreatment with the RIPK1 inhibitor Nec1s or the We firstexaminedthe expression of RIPK3and MLKL in MLKL inhibitor necrosulfamide (NSA) inhibited this human neutrophils, peripheral blood mononuclear cells process (Fig. 7a, b). Furthermore, as a second and more (PBMCs), and platelets by immunofluorescence staining physiological inducer thrombin triggered the same as well as immunoblotting. Immunostaining revealed interaction of granulocytes and platelets, which was that neutrophils and platelets both express RIPK3 and rescued by Nec1s and NSA pretreatment (Fig. 7c). MLKL (Supplemental Fig. 3a, b). In addition, immuno- Finally, we examined whether platelets themselves blotting analysis showed the presence of RIPK3 and undergo necroptosis by thrombin stimulation. The Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 5 of 11 Fig. 3 Nec1s suppresses necroptosis and NET-related signaling pathway during thrombus formation. a Representative figures of IVC-ligated mice with pretreatment of vehicle and b Nec1s. From left panel, the staining shows TUNEL, Ly6b, RIPK3, MLKL, CitH3, and F4/80. Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm). c Quantification of positive area of each staining. Data are mean ± SEM in each group. *p < 0.05 vs. respective control platelet death and activation in vitro was examined by Discussion flow cytometry using annexin V and P-selectin as acti- We had hypothesized that MLKL-dependent neutrophil vation markers, respectively. TNFα/zVAD-stimulated necroptosis would contribute to VTE and employed specific human platelets up-regulated annexin V and P-selectin; antagonists and Mlkl-deficient mice to address this concept however, these phenomena were not inhibited by experimentally both in vitro and in vivo. We found the Nec1s and NSA (Supplemental Fig. 4a). Similarly, essential mediators of necroptosis, RIPK3 and MLKL, to be thrombin up-regulated annexin V and P-selectin present in IVC thrombi of humans and mice. Interfering with expression in platelets, whereas Nec1s or NSA had no necroptosis, either with the specific antagonist Nec1s or with inhibitory effect (Supplemental Fig. 4b). Taken together, genetic deletion of Mlkl, partially protected mice from IVC activated platelets induce neutrophil necroptosis and ligation-induced venous thrombosis. Mechanistically, neutrophil–platelet aggregation. thrombin-related platelet activation triggered neutrophil Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 6 of 11 Fig. 4 Genetic depletion of Mlkl reduces thrombus size in the IVC ligation model. a Macroscopic findings of thrombi in IVC-ligated wild-type −/− −/− (left) and Mlkl mice. Scale bar = 1 mm. b The graph shows the thrombus size of IVC-ligated wild-type (n = 7) and Mlkl mice (n = 7). Data are mean ± SEM in each group. *p < 0.05 vs. respective control death and neutrophil-platelet aggregation, which both could numerous other crystals and microparticles of different be reversed by specific necroptosis inhibitors. Platelet acti- sizes and shapes induce neutrophil necroptosis via RIPK1, 14,15 vation itself did not involve this pathway. We, therefore, RIPK3, and MLKL . Neutrophil necroptosis has also conclude that in VTE activated platelets induce neutrophil been observed in other conditions including exposure to necroptosis, a process generating the release of chromatin granulocyte–macrophage colony-stimulating factor fol- and DAMPs that contribute to clot formation (Fig. 8). lowed by the ligation of adhesion receptors such as CD44, NETs form predominantly during the organizing stage of CD11b, CD18, or CD15 . Our data add thrombin-activated VTE development . NETs are released by neutrophils platelets to the potential triggers of neutrophil necroptosis, present in the lesion, and several studies identified platelet- although the precise outside-in signaling mechanism 9–11,23 derived HMGB1 as a trigger for NET formation remains to be determined. We also do not claim that the .Lytic proteases, histones, and DAMPs released along with NETs process of neutrophil necroptosis is identical to that of NET certainly contribute to the local and systemic inflammation release or what has been called “NETosis.” However, the 2,8,24,25 associated with VTE . However, the sticky DNA itself consequence of plasma cell rupture in neutrophil necrosis is seems to be an essential component of the clot and the same, that is, sticky NET-like chromatin immobilizes synergizes with the fibrin mesh to retain red blood cells . adjacent particles, which in VTE are red blood cells, pla- Indeed, endogenous DNases counterbalance this phenom- telets, and the fibrinogen mesh . enon in conceptually similar manner to plasmin that Interestingly, we found that platelets also express RIPK3 degrades the fibrin mesh . There is an ongoing debate and MLKL. These are ubiquitous cytoplasmic proteins, whether NET release is necessarily associated with neu- which are obviously shed from megakaryocytes during trophil death or not. During host defense neutrophils have platelet formation. Although platelet necrosis is known to been shown to continue migrating also after NET release, be regulated by mitochondrial effect with calcium reflux 16,27 31 but this has not been observed in other disease settings . and ATP depletion , how necroptosis contributes to the For example, in gout the exposure to urate crystals triggers platelet death remains unknown. It has been reported that crystal–NET aggregates involving lytic neutrophil death and deletion of RIPK3 from megakaryocytes and platelets causes production of a sticky creamy mass of NETs, dead a marked defect in platelet aggregation and attenuates 28,29 neutrophils, and crystals, called the gouty tophus .In dense granule secretion in response to thrombin or a VTE the process and results are conceptually similar, thromboxane A2 analog in vitro, and delay vascular although the additional presence of blood components such occlusion time in a mouse model of arterial thrombosis .It as platelets, red blood cells, and the fibrin mesh produce a should be noted that RIPK3 has promiscuous biological much higher consistency of clots vs. gouty tophi. Never- functions beyond necroptosis, for example, in apoptosis or theless, neutrophil death is essential in this process and interleukin-1-dependent or nuclear factor-κB-dependent 12 13 contributes to vascular occlusion, obstructing the blood inflammation , as well as platelet activation .Never- flow. Indeed, the interesting thing in the setting of VTE is theless, we did not find any evidence that MLKL inhibition that neutrophils undergo necroptosis, a form of regulated affects thrombin-induced platelet activation. Therefore, we cell necrosis . We recently described that urate as well as consider the VTE phenotype of Mlkl-deficient mice largely Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 7 of 11 Fig. 5 Mlkl gene deficiency suppresses necroptosis and NET-related signaling pathways during thrombus formation. From the left panel, −/− TUNEL, Ly6b, RIPK3, CitH3, and F4/80 staining. a Representative figures of IVC-ligated wild-type mice and b IVC-ligated Mlkl mice (scale bar = 1 mm). Upper figures show the whole thrombus (scale bar = 1 mm) and lower figures show magnified image (scale bar = 250 μm). c Quantification of positive area of each staining. Data are mean ± SEM in each group. *p < 0.05 vs. respective control relate to the lack of neutrophil necroptosis. Unfortunately, necroptosis may interfere with clotting, which might be flox Mlkl mice were not accessible to us to study cell-type- explored for therapeutic purposes. specificdeletionofMLKL. In summary, in VTE activated platelets, and possibly Materials and methods other triggers, induce neutrophil necroptosis, a process Venous thrombosis model generating the release of chromatin and DAMPs One hundred percent flow obstruction of the IVC was that contribute to clot formation. Thus, inhibitors of induced in 12- to 13-week-old male wild-type C57BL/6N Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 8 of 11 Fig. 6 Activated platelets stimulate neutrophils leading to up-regulation of necroptosis-related and NET-related signaling molecules in vitro. a Upper, middle, and lower panel show co-culture images of neutrophils with non-activated platelets, neutrophils with thrombin-activated platelets, and Nec1s-treated neutrophils with thrombin-activated platelets. Immunofluorescent images show neutrophil elastase (NE): green; citrullinated histones H3 (CitH3): red; RIPK3: red; and MLKL: red. Scale bar = 50 μm. Quantification of CitH3 (b), RIPK3 (c), MLKL (d) area and LDH release of supernatant (e)in unstimulated neutrophils, thrombin-treated neutrophils, non-activated platelets, vehicle-treated neutrophils with thrombin-activated platelets, and Nec1s-treated neutrophils with thrombin-activated platelets. Data represent the mean ± SEM of three independent experiments and were analyzed using the paired t test. *p < 0.05 vs. respective control; **p < 0.01 vs. respective control mice (Charles River Laboratories, Sulzfeld, Germany) or sutures. Anesthesia was antagonized by subcutaneous −/− Mlkl mice under the maintenance of normal body injection of atipamezol 2.5 mg/kg and flumazenil 0.5 mg/ temperature by employing preoperative heat supply and kg and pain control was assured by regular subcutaneous online core body temperature recording as described . injections of buprenorphine 1 mg/kg every 8 h. Mice with Mice were anesthetized by intraperitoneal injection of surgical complications such as bleeding or injury of the medetomidine (0.5 mg/kg), midazolam (5 mg/kg), and IVC were excluded because these factors could possibly fentanyl (0.05 mg/kg) before median laparotomy was affect thrombus formation. Other groups of C57BL/6N performed to carefully expose and completely ligate the wild-type mice were treated with Nec1s (1.65 mg/kg, IVC using 7-0 prolene (ETHICON) exactly below the left intraperitoneally, Bio Vision, USA) or vehicle (10% renal vein without manipulating side branches. After dimethyl sulfoxide (DMSO) in phosphate-buffered saline ligation, the abdominal wall and skin were closed by (PBS)) 1 h before the surgery. All mice were sacrificed Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 9 of 11 Fig. 7 Nec1s and necrosulfonamide (NSA) inhibit aggregation between neutrophils and platelets in vitro. a Human whole blood was stimulated with TNFα/zVAD in the presence of vehicle, Nec1s, and NSA. Upper flow cytometry images show the platelet population gated by forward scatter (FSC)/sideward scatter (SCC). Lower images show neutrophil–platelet aggregates by CD61/CD66 gating and the aggregation ratio in TNFα/ zVAD-treated (b) and thrombin-treated (c) blood. Data represent the mean ± SEM of three independent experiments and were analyzed using the paired t test. *p < 0.05 vs. respective control 3 days after surgery and thrombus weight (without ves- Germany) was used to detect dying cells inside the sels) was measured as a primary endpoint of clot forma- thrombus following the manufacturer’s description. Positive tion. As a bleeding test 10- to 12-week-old male C57BL/ cells were quantified using the ImageJ software. −/− 6N wild-type or Mlkl mice were anesthetized using isofluorane. A 2 mm segment of the tail tip was cut Histological examination in human thrombus using a scalpel, and the tail was put in 37 °C PBS . Paraffin-embedded sections of a human IVC thrombus Bleeding time was recorded up to when bleeding had from a patient with renal cell carcinoma were stained with completely stopped. All animal-related procedures fulfilled H&E and immunohistochemistry was performed using the directive 2010/63/EU and were optimized in terms of the following primary antibodies rabbit anti-CD61 3R recommendations and approved by the local govern- (LifeSpan Biosciences, Inc. Seattle, WA, USA), rabbit mental authorities (ROB-55.2Vet-2532.Vet_02-17-54). anti-fibrinogen, rabbit anti-myeloperoxidase, rabbit anti- RIPK3, rabbit anti-phosphorylated MLKL (all from Histological examination Abcam, Cambridge, UK). Signal detected was performed Thrombi were embedded in paraffinand 3 μmsections using routine procedures as described . were deparaffinized and rehydrated as previously descri- bed . Sections were stained with H&E or prepared for In vitro experiments immunohistochemistry. A 0.3% H O was used for inhibi- Blood was obtained from healthy donors after providing 2 2 tion of endogenous peroxidase. Primary antibodies included written informed consent on forms approved by the ratanti-mouseLy6b(neutrophils, AbDSerotec,Oxford, “Ethikkommission der Medizinischen Fakultät der LMU” UK), rabbit anti-CitH3 (netting neutrophils, Abcam, Cam- and all experiments were performed in accordance with bridge, UK), rabbit anti-mouse RIPK3 (Abcam, Cambridge, their guidelines and regulations. Neutrophils were isolated UK), anti-mouse MLKL (kindly provided by Andreas Lin- using standard dextran sedimentation followed by 14,15 kermann, Dresden), and rat anti-mouse F4/80 (Serotec, Ficoll–Hypaque density centrifugation procedures . Oxford, UK) . TUNEL staining kit (Roche, Mannheim, For platelet isolation, blood was collected into sodium Official journal of the Cell Death Differentiation Association Nakazawa et al. Cell Death Discovery (2019) 5:6 Page 10 of 11 Fig. 8 Schema of activated platelet-induced neutrophil necroptosis in DVT. During thrombus formation, RBCs initiate thrombin generation and platelet activation. The activated platelets affect neutrophils to induce neutrophil necroptosis via the phosphorylation of MLKL. Necroptotic neutrophil-derived extracellular chromatin can interact with fibrin mesh and activate endothelial cells, resulting in the aggravation of rigid clot formation as immunothrombosis. RBC red blood cell, pMLKL phosphorylated mixed lineage kinase domain-like citrate-coated tubes. Platelet-rich plasma was obtained by CD11b (BD Biosciences), and APC-CD45 (BioLegend) antibodies were used to identify circulating neutrophils centrifugation (200 rpm, 20 min). Neutrophils were sus- pended in RPMI (5 × 10 cells/well), and seeded onto and activated monocytes in peripheral blood. Mouse either eight-well micro-slides (Ibidi, Martinsried, Ger- plasma was analyzed for histone–nucleosome complexes many) or 96-well plates, and incubated in a 5% carbon by ELISA (Roche). Whole blood was analyzed by flow dioxide atmosphere at 37 °C for 30 min. Neutrophils were cytometry to quantify circulating immune cells . In vitro, pre-treated with Nec1s (100 µM, Enzo, Lörrach, Ger- human platelets and whole blood were used. Platelets many) or vehicle (1% DMSO in PBS) for 30 min and then were gated by APC-CD42b (BioLegend) and platelet stimulated with thrombin (0.05 U/ml, Merck Millipore, activation and death were determined by PerCP-CD62p Darmstadt, Germany), non-activated platelets, and (BioLegend) and FITC-Annexin V (BD Pharmingen), thrombin (0.05 U/ml for 3 min) activated platelets (1 × 10 respectively. Platelet–granulocyte aggregation was deter- cells/well). After 3-h incubation, the microslides were mined using anti-human FITC-CD61 and PE-CD66 fixed with 4% paraformaldehyde (PFA) and analyzed for (BioLegend) antibody in accordance with the manu- CitH3, RIPK3, and MLKL expression by immuno- facturer’s instructions. fluorescence staining. In 96 plates, neutrophil death was quantified by the LDH assay (Sigma Aldrich, Steinheim, Histone-nucleosome assay Germany) using neutrophil supernatants. To induce Serum histone was evaluated by histone–DNA com- neutrophil–platelet aggregation, human whole blood was plexes ELISA kit (Roche, Mannheim, Germany). stimulated by either the combination TNFα (200 ng/ml) and zVAD (20 µM) or 0.05 U/ml thrombin (for 10 min) Immunoblotting with or without pretreatment of Nec1s (100 µM) and Blood cells were also analyzed by standard immuno- necrosulfonamide (10 µM, Calbiochem). blotting. Cell pellets were lysed with RIPA buffer (Sigma, USA), the extracted proteins were separated by sodium Flow cytometric analysis dodecyl sulfate-polyacrylamide gel electrophoresis, and Flow cytometric analysis was performed on a FACS transferred to a polyvinylidene difluoride membrane. Calibur flow cytometer (BD Biosciences). In mouse Anti-β-actin, RIPK3, and MLKL antibodies (Abcam, UK) experiments, anti-mouse FITC-Ly6G, PerCP-Ly6C, PE- were used for detection of molecules expression. Official journal of the Cell Death Differentiation Association Nakazawa et al. 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